From the natural sciences, engineering and medicine, microscopes for capturing image information from different types of specimen are known in numerous technical variants. To an increasing degree, coherent light sources, which include one or a plurality of lasers, for example, are being used as light sources for producing excitation beams. In particular, laser microscopes can be designed as scanning microscopes.
Scanning microscopes are known in diverse variants that differ, for example, in the type and generation of the microscope beam. Thus, for example, electromagnetic beams can be used in the optical, infrared or ultraviolet region of the spectrum. Other distinctions among the various types of scanning microscopes are evident in the interaction of the microscope beam(s) with the specimen to be examined. In the following description, reference is made first and foremost to fluorescence microscopes, where the microscope beam, respectively excitation beam excites the fluorescence of a specimen that can then be captured and used for image acquisition. Apart from that, there are numerous other measuring principles, however, such as those based on spectroscopic laser methods, those based on particle emissions, or other measuring principles. The present invention described in the following can be applied, in principle, to all such methods and designs, as well as to other microscopes whose functioning is not based on the scanning principle.
Supplying excitation light having one or a plurality of predefined wavelengths presents a significant challenge to numerous known microscopes, regardless of the method used. In this context, depending on the type of microscopy method and/or the type of specimen (for example, the specimen quality or the particular dye used to stain the specimen), one or a plurality of excitation light beams are needed, which typically must have predefined spectral properties.
When working with conventional laser microscopes, the excitation light is supplied by one or a plurality of excitation lasers; typically, however, merely a limited wavelength region, respectively a limited selection of spectral lines being available. As a result, the microscopes are limited in their application to certain specimen types, specific microscopy methods, and/or to specific dyes used in staining the specimen. In many cases, this limited application spectrum is not satisfactory.
For that reason, microscopes are known which are able to utilize coherent white light sources as a light source. These microscopes also use broadband coherent light sources, similarly to conventional microscopes having non-coherent light sources that generate incoherent light having a broad wavelength distribution, from which the requisite spectral regions are then selected using wavelength-selective elements. These types of light sources, which have a broad wavelength spectrum, are also commonly referred to as “white light sources.”
Light sources, whose light has a broad wavelength spectrum, can be realized by the incoupling of laser radiation, particularly with the aid of special optical elements, such as a tapered (i.e., structured, particularly in terms of its index of refraction) fiber, a microstructured fiber (in particular, a photonic crystal fiber, PCF), a holey fiber, a photonic bandgap fiber (PBC) or a specially doped fiber (for example, a fiber having a rare-earth-metal of semiconductor doping), for example. These types of white-light lasers are often referred to as supercontinuum white-light lasers. Examples of such broadband white-light lasers are given in the German Patent Applications DE 101 15 488 A1, DE 101 15 509 A1 or DE 101 15 589 A1. The present invention described in the following is based on these white-light lasers, but is also well suited for use with other coherent broadband light sources.
From the broadband coherent light produced by the white-light lasers or broadband light sources, a specific wavelength or a specific spectral region can be subsequently selected with the aid of wavelength-selective elements. Various wavelength-selective elements of this kind, such as prisms or gratings, have been known for quite some time.
In recent years, however, wavelength-selective elements that are based on the acousto-optical effect (acousto-optical elements) are being used to an increasing degree. These types of acousto-optical elements typically have what is generally known as an acousto-optical crystal (for example, a tellurium dioxide crystal, TeO2), which is cut in a suitable crystal direction. Mounted on this acousto-optical crystal are one or a plurality of acoustic signal transmitters, which are also referred to as “transducers.” A transducer of this kind typically has a piezoelectric material, as well as two or more electrodes contacting this material. By applying radio frequencies, typically within the range of between 30 MHz and 800 MHz, to the electrodes, the piezoelectric material is excited to oscillate, thereby enabling an acoustic wave to be produced that propagates through the crystal. After propagating through an optical interaction region, this acoustic wave is mostly absorbed or reflected at the opposite crystal side. A distinguishing feature of acousto-optical crystals is that the sound wave produced alters the optical properties of the crystal, an optical grating or a comparable optically active structure (hologram) being induced by the sound. Light passing through the crystal can ultimately undergo diffraction at this optical grating and be deflected into different diffraction orders or diffraction directions.
In the case of acousto-optical components, one distinguishes between these components which influence the entire incident light to a greater or lesser degree independently of the wavelength (for example, acousto-optical modulators) and those components which act selectively on individual wavelengths (for example, as a function of the radio frequency irradiation) (acousto-optical tunable filters, AOTF). In many cases, the acousto-optical elements have double-refractive crystals, such as the mentioned tellurium dioxide, for example, the position of the crystal axis relative to the plane of incidence of the light and the polarization thereof determining the optical properties of the acousto-optical element.
Thus, with the aid of the mentioned acousto-optical filters (AOTFs), one or a plurality of wavelength regions can be selected from the wavelength spectrum of the white light source. An incident light beam, which propagates through the phase grating in the crystal, is then split into its diffraction orders. By varying the frequency f0 the acoustic wave, the frequency of the phase grating in the acousto-optical crystal changes, and thus also the wavelength λ0 of the diffracted light. AOTFs can be implemented in such a way that the centroid wavelengths λ0 of the selected wavelength regions exit the acousto-optical crystal colinearly. However, other wavelengths within the selected wavelength regions have a different direction of radiation.
This change in the direction of radiation, respectively the spatial separation between the desired wavelength (in the following, also referred to as target wavelength) λ0 and the remaining light that is radiated into the acousto-optical crystal is utilized to separate the light. This is likewise discussed, for example, in the already cited German Patent Application DE 101 15 488 A1 which describes a light source having a white-light laser (including a fiber) and a downstream AOTF.
However, a difficulty encountered when working with the known acousto-optical filters is that, in practice, there is not a unique correlation between an incoupled radio frequency of one acoustic wave and a specific target wavelength. The shape of the transfer function of the AOTF, thus the frequencies, respectively wavelengths of the target light beam which is transmitted through the AOTF given a fixed radio frequency, does not represent an idealized δ function, but rather corresponds approximately to the following function:T˜sin2(f−f0)/(f−f0)2  (1)
This means that the transfer function of an AOTF has numerous secondary maxima, which may be considerably less pronounced than the central principal maximum at the frequency f0, respectively the wavelength λ0 of the light, but can have the effect of interfering with the spectroscopy, however.
Thus, for example, light of the target wavelength (principal maximum of the transfer function of the AOTF) is superimposed with light in the region of the secondary maxima since the white light source likewise emits in this spectral region and the AOTF is transmissive in this region. This light later superimposes itself on the actual detection light, for example, after being reflected at the specimen. However, wavelength-selective elements, which are supposed to separate the actual excitation light from the detection light, are often so highly wavelength-selective that they merely separate the actual detection light (for example, fluorescent light of the specimen) from a specific excitation wavelength λ0, but do not ensure an adequate separation in the case of excitation light outside of the wavelength λ0. This can lead to excitation light reaching the detector which, in turn, seriously degrades the signal-to-noise ratio of the specimen image. Thus, in the case of a fluorescence spectroscopy, the actual fluorescence signals, in particular, can be weaker by orders of magnitude than the excitation light, so that the actual signal is seriously degraded by the excitation light that also reaches the detector.
This difficulty is especially evident when working with microscopes where excitation light and detection light are separated with the aid of acousto-optical beam splitters, AOBS. An AOBS also has a transfer function where the separating action is characterized by a peak maximum in the transfer function. Typically, however, this principal maximum is substantially broader than the maximum of the transfer function of an AOTF, so that one or a plurality of secondary maxima of the transfer function of the AOTF fall within the maximum of the transfer function of the AOBS. This means that the AOBS allows spectral components of the white light source, which reside within the region of these secondary maxima of the transfer function of the AOTF, to reach the detector of the microscope to a large degree.